Therapeutic methods and compounds

ABSTRACT

The invention provides diketopiperazines of formula I. The invention also provides pharmaceutical compositions comprising the diketopiperazines, or pharmaceutically-acceptable salts or prodrugs thereof, as the active ingredient. The invention further provides therapeutic treatments that utilize the diketopiperazines of formula I, including inhibition of a proliferative disease or condition, inhibition of angiogenesis, treatment of an angiogenic disease or condition, treatment of cancer and precancerous conditions, treatment of a fibrotic disorder, treatment of a viral infection, treatment of an Akt-mediated disease or condition, inhibition of the production, release or both of matrix metalloproteinase-9, and inhibition of Akt activation.

This application claims the benefit of provisional application No.61/056,379, filed May 27, 2008, the complete disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to diketopiperazines and pharmaceuticalcompositions comprising them as the active ingredient. The inventionalso relates to the therapeutic treatments that utilize thediketopiperazines, including inhibition of a proliferative disease orcondition, inhibition of angiogenesis, treatment of an angiogenicdisease or condition, treatment of cancer and precancerous conditions,treatment of a fibrotic disorder, treatment of a viral infection,inhibition of Akt activation, treatment of an Akt-mediated disease orcondition, and inhibition of the production, release or both of matrixmetalloproteinase-9.

BACKGROUND

Diketopiperazines have been reported to exhibit a variety of biologicalactivities. See, e.g., U.S. Pat. No. 3,941,790 (cancer treatment), U.S.Pat. No. 4,289,759 (immunoregulatory agents), U.S. Pat. No. 4,331,595(immunoregulatory agents), U.S. Pat. No. 4,940,709 (platelet activatingfactor (PAF) antagonists), U.S. Pat. No. 5,700,804 (inhibitors ofplasminogen activator inhibitor), U.S. Pat. No. 5,750,530 (inhibitors ofplasminogen activator inhibitor), U.S. Pat. No. 5,990,112 (inhibitors ofmetalloproteases), U.S. Pat. No. 6,537,964 (chemosensitizing reversalagents for treatment of multiple drug resistant cancers), U.S. Pat. No.6,555,543 (inhibitors of PAF, the production and/or release ofinterleukin 8 (IL-8) and inflammation), and U.S. Pat. No. 6,815,214(treatment of inflammatory conditions associated with, e.g., cancer andasthma), PCT applications WO 97/36888 (inhibitors of farnesyl-proteintransferase), WO 98/9968 (treatment for infections, cancer and othermalignant diseases), WO 99/40931 (treatment of central nervous systeminjury), and WO 04/87162 (agents for treatment of drug resistantcancer), EP application 43219 (immunoregulatory agents), Japaneseapplication 63 290868 (PAF antagonists), Japanese application 31 76478(immunosuppressive agents), Japanese application 51 63148(anti-neoplastic agents), Shimazaki et al., Chem. Pharm. Bull., 35(8),3527-3530 (1987) (PAF antagonists), Shimazaki et al., J. Med. Chem., 30,1709-1711 (1987) (PAF antagonists), Shimazaki et al., Lipids, 26(12),1175-1178 (1991) (PAF antagonists), Yoshida et al., Prog. Biochem.Pharmacol., 22, 68-80 (1988) (PAF antagonists), Alvarez et al., J.Antibiotics, 47(11), 1195-1201 (1994) (inhibitors of calpain).

SUMMARY OF THE INVENTION

The present invention provides a diketopiperazine having the followingformula:

wherein:

-   -   R¹ is:    -   (a) a side chain of an amino acid, wherein the amino acid is        glycine, alanine, valine, norvaline, α-aminoisobutyric acid,        2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,        isoleucine, norleucine, serine, homoserine, threonine, aspartic        acid, asparagine, glutamic acid, glutamine, lysine,        hydroxylysine, histidine, arginine, homoarginine, citrulline,        phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,        thyroxine, cysteine, homocysteine, methionine, penicillamine or        ornithine;    -   (b) —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with the        adjacent ring nitrogen forms proline or hydroxyproline; or    -   (c) a derivative of a side chain of an amino acid, wherein the        amino acid is one of those recited in (a), and the derivatized        side chain has:        -   (i) an —NH₂ group replaced by an —NHR³ or —N(R³)₂ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (ii) an —OH group replaced by an —O—PO₃H₂ or —OR³ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (iii) a —COOH group replaced by a —COOR³ group, wherein each            R³ may independently be a substituted or unsubstituted            alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,            arylalkyl or heteroaryl;        -   (iv) a —COOH group replaced by a —CON(R⁴)₂ group, wherein            each R⁴ may independently be H or a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (v) an —SH group replaced by —S—S—CH₂—CH(NH₂)—COOH or            —S—S—CH₂—CH₂—CH(NH₂)—COOH;        -   (vi) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)—            group;        -   (vii) a —CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH            group; and/or        -   (viii) an H which is attached to a carbon atom replaced by a            halogen;    -   R² has formula II, III or IV:

wherein:

each R⁵ is independently aryl, heteroaryl, alkyl, cycloalkyl,heterocycloalkyl, alkoxy, aryloxy, acyl, carboxyl, hydroxyl, halogen,amino, nitro, sulfo or sulfhydryl, wherein each alkyl is optionallysubstituted with hydroxyl, amino or sulfhydryl;

n is from 0 to 5; and

R⁶ is hydrogen or lower alkyl.

The invention also provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier and an active ingredient, whereinthe active ingredient is a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

The invention further provides a method of treating a proliferativedisease or condition. The method comprises administering to an animal inneed thereof an effective amount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

In addition, the invention provides a method of inhibiting angiogenesis.The method comprises administering to an animal in need thereof aneffective amount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

The invention further provides a method of treating an angiogenicdisease or condition. The method comprises administering to an animal inneed thereof an effective amount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

In addition, the invention provides a method of treating a cancer. Themethod comprises administering to an animal in need thereof an effectiveamount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

The invention further provides a method of treating a precancerouscondition. The method comprises administering to an animal in needthereof an effective amount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

The invention also provides a method of treating a fibrotic disorder.The method comprises administering to an animal in need thereof aneffective amount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

In addition, the invention provides a method of treating a viralinfection. The method comprises administering to an animal in needthereof an effective amount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

The invention also provides a method of inhibiting the production,release or both of matrix metalloproteinase-9 (MMP-9) by cells. Themethod comprises contacting the cells with an effective amount of adiketopiperazine of formula I or a pharmaceutically-acceptable salt orprodrug thereof.

In addition, the invention provides a method of inhibiting theactivation (phosphorylation) of Akt by cells. The method comprisescontacting the cells with an effective amount of a diketopiperazine offormula I or a pharmaceutically-acceptable salt or prodrug thereof.

The invention further provides a method of treating an Akt-mediateddisease or condition. The method comprises administering to an animal inneed thereof an effective amount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Schematic illustrating the synthesis ofbiphenyl-4-yl-(3,6-dioxo-piperazin-2-yl)-acetic acid methyl ester (Cpd.5).

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS OF THEINVENTION

The present invention provides diketopiperazines which have thefollowing formula I:

wherein:

-   -   R¹ is:    -   (a) a side chain of an amino acid, wherein the amino acid is        glycine, alanine, valine, norvaline, α-aminoisobutyric acid,        2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,        isoleucine, norleucine, serine, homoserine, threonine, aspartic        acid, asparagine, glutamic acid, glutamine, lysine,        hydroxylysine, histidine, arginine, homoarginine, citrulline,        phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,        thyroxine, cysteine, homocysteine, methionine, penicillamine or        ornithine;    -   (b) —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with the        adjacent ring nitrogen forms proline or hydroxyproline; or    -   (c) a derivative of a side chain of an amino acid, wherein the        amino acid is one of those recited in (a), and the derivatized        side chain has:        -   (i) an —NH₂ group replaced by an —NHR³ or —N(R³)₂ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (ii) an —OH group replaced by an —O—PO₃H₂ or —OR³ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (iii) a —COOH group replaced by a —COOR³ group, wherein each            R³ may independently be a substituted or unsubstituted            alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,            arylalkyl or heteroaryl;        -   (iv) a —COOH group replaced by a —CON(R⁴)₂ group, wherein            each R⁴ may independently be H or a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (v) an —SH group replaced by —S—S—CH₂—CH(NH₂)—COOH or            —S—S—CH₂—CH₂—CH(NH₂)—COOH;        -   (vi) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)—            group;        -   (vii) a —CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH            group; and/or        -   (viii) an H which is attached to a carbon atom replaced by a            halogen;

R² has formula II, III or IV:

wherein:

each R⁵ is independently aryl, heteroaryl, alkyl, cycloalkyl,heterocycloalkyl, alkoxy, aryloxy, acyl, carboxyl, hydroxyl, halogen,amino, nitro, sulfo or sylfhydryl, wherein each alkyl is optionallysubstituted with hydroxyl, amino or sulfhydryl;

n is from 0 to 5; and

R⁶ is hydrogen or lower alkyl.

Preferably, R¹ is a side chain of glycine, alanine, valine, norvaline,α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyricacid, leucine, isoleucine, norleucine, serine, homoserine, threonine,aspartic acid, asparagine, glutamic acid, glutamine, lysine,hydroxylysine, histidine, arginine, homoarginine, citrulline,phenylalanine, p-aminophenylalanine, tyrosine, tryptophan, thyroxine orornithine, or is a derivative of one of these side chains.

More preferably, R¹ is a side chain of glycine, alanine, valine,norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid,2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine,homoserine, threonine, aspartic acid, asparagine, glutamic acid,glutamine, lysine, hydroxylysine, arginine, homoarginine, citrulline orornithine, or is a derivative of one of these chains.

Even more preferably, R¹ is the side chain of glycine, alanine, valine,leucine or isoleucine, more preferably glycine or alanine, mostpreferably glycine.

Preferably, R² has formula II or III, most preferably II.

R⁵ is preferably aryl, heteroaryl or aryloxy. More preferably R⁵ is arylor aryloxy. Most preferably R⁵ is phenyl or phenoxy.

Preferably, n is 1-3. Most preferably n is 1. When n is 1, R⁵ ispreferably in the 4 (para) position on the ring.

R⁶ is preferably methyl.

The most highly preferred compound isbiphenyl-4-yl-(3,6-dioxo-piperazin-2-yl)-acetic acid methyl ester(referred to as Cpd. 5 herein).

By “replaced” is meant that, with reference to the formula of an aminoacid side chain, the specified group is replaced by the other specifiedgroup. For instance, the formula of the isoleucine side chain is—CH(CH₃)—CH₂—CH₃. If the terminal —CH₃ group is replaced with a —CH₂—OHgroup, then the formula of the resulting derivatized isoleucine sidechain would be —CH(CH₃)—CH₂—CH₂—OH. As another example, the formula ofthe alanine side chain is —CH₃. If one of the hydrogen atoms is replacedby a chlorine atom, then the resulting derivatized alanine side chainwould be —CH₂—Cl. Note that the side chain of glycine is —H and, if thisH is replaced by a chlorine (or other halogen) atom, the resulting sidechain will —Cl, with the chlorine atom attached to the ring carbon(e.g., R¹═—Cl)

By “side chain” of an amino acid is meant that portion of the amino acidattached to the common NH₂—CH—COOH backbone of all of the amino acidslisted above. For instance, the side chain of glycine is —H, the sidechain of alanine is —CH₃, and the side chain of serine is —CH₂OH.

By “acyl” is meant a moiety of the formula —C(O)R⁷, wherein R⁷ ishydrogen, alkyl, cycloalkyl or aryl.

By “alkoxy” is meant a moiety of the formula —OR⁸, wherein R⁸ is alkyl.An example of an alkoxy group is methoxy (—O—CH₃).

By “alkyl” is meant a monovalent saturated straight-chain or branchedhydrocarbon containing 1-10 carbon atoms, preferably 1-8, carbon atoms.Each alkyl may, optionally, be substituted with one or more amino,hydroxyl or sulfhydryl groups. “Lower alkyl” means a monovalentsaturated straight-chain or branched hydrocarbon containing 1-6 carbonatoms.

By “alkylaryl” is meant a lower alkyl having an H replaced by an aryl(e.g., —CH₂—C₆H₅ or —CH₃CH(C₆H₅)CH₃).

By “amino” is meant a moiety of the formula —NR⁹R¹⁰, wherein each R⁹ andR¹⁰ is independently H or lower alkyl.

By “aryl” is meant a monovalent mono-, bi- or tricyclic aromatichydrocarbon moiety having 6-14 ring carbon atoms. Preferred is phenyl.

By “aryloxy” is meant a moiety of the formula —OR¹¹ wherein R¹¹ is anaryl. Preferred is phenoxy.

By “arylalkyl” is meant an aryl having an H replaced by a lower alkyl(e.g., —C₆H₄—CH₃).

By “carboxyl” is meant —COOH.

By “cycloalkyl” is meant a saturated monovalent mono- or bicyclichydrocarbon moiety having three to ten ring carbon atoms. Preferably,the cycloalkyl contains 4-8 ring carbon atoms. The most preferredcycloalkyl is cyclohexyl.

By “halogen” is meant chlorine, fluorine, bromine or iodine. Preferredis chlorine or bromine.

By “heteroaryl” is meant an aryl having at least one, preferably no morethan three, of the ring carbon atoms replaced by an O, S or N.

By “heterocycloalkyl” is meant a cycloalkyl having at least one,preferably no more than three, of the ring carbon atoms replaced by anO, S or N.

By “hydroxyl” is meant —OH.

By “nitro” is meant —NO₂.

By “substituted” is meant that the moiety is substituted with one ormore substituents selected from the following group: —OH, NH₂, —SH,—COOH and/or a halogen atom.

By “sulfhydryl” is meant —SH.

By “sulfo” is meant —SO₃H or SO₂.

Methods of making diketopiperazines are well known in the art, and thesemethods may be employed to synthesize the diketopiperazines of theinvention. See, e.g., U.S. Pat. Nos. 5,817,751, 5,932,579, 5,990,112,6,395,774, 6,555,543 and 7,288,345, US Patent Application PublicationsNumbers 2004/00132738 and 2004/0024180, PCT applications WO 96/00391 andWO 97/48685, Smith et al., Bioorg. Med. Chem. Letters, 8, 2369-2374(1998), Prakash et al., Bioorg. Med. Chem. Letters, 10, 3034-3048(2002), Fischer, J. Peptide Sci., 9, 9-35 (2003), and Zeng et al.,Bioorg. Med. Chem. Letters, 15, 3034-3038 (2005). The completedisclosures of these references are incorporated herein by reference asexemplary methods of synthesizing diketopiperazines of the presentinvention.

For instance, diketopiperazines can be prepared by first synthesizingdipeptides. The dipeptides can be synthesized by methods well known inthe art using L-amino acids, D-amino acids or a combination of D- andL-amino acids. Preferred are solid-phase peptide synthetic methods. Ofcourse, dipeptides are also available commercially from numeroussources, including DMI Synthesis Ltd., Cardiff, UK (custom synthesis),Sigma-Aldrich, St. Louis, Mo. (primarily custom synthesis), PhoenixPharmaceuticals, Inc., Belmont, Calif. (custom synthesis), FisherScientific (custom synthesis) and Advanced ChemTech, Louisville, Ky.Once the dipeptide is synthesized or purchased, it is cyclized to form adiketopiperazine. This can be accomplished by a variety of techniques.

For example, U.S. Patent Application Publication Number 2004/0024180describes a method of cyclizing dipeptides. Briefly, the dipeptide isheated in an organic solvent while removing water by distillation.Preferably, the organic solvent is a low-boiling azeotrope with water,such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol,2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride,chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal,dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone,3-pentanol, toluene and xylene. The temperature depends on the reactionspeed at which the cyclization takes place and on the type ofazeotroping agent used. The reaction is preferably carried out at50-200° C., more preferably 80-150° C. The pH range in which cyclizationtakes place can be easily determined by the person skilled in the art.It will advantageously be pH 2-9, preferably pH 3-7.

When one or both of the amino acids of the dipeptide has, or isderivatized to have, a carboxyl group on its side chain (e.g., asparticacid or glutamic acid), the dipeptide is preferably cyclized asdescribed in U.S. Pat. No. 6,555,543. Briefly, the dipeptide, with theside-chain carboxyl still protected, is heated under neutral conditions.Typically, the dipeptide will be heated at from about 80° C. to about180° C., preferably at about 120° C. The solvent will be a neutralsolvent. For instance, the solvent may comprise an alcohol (such asbutanol, methanol, ethanol, and higher alcohols, but not phenol) and anazeotropic co-solvent (such as toluene, benzene, or xylene). Preferably,the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. Theheating is continued until the reaction is complete, and such times canbe determined empirically. Typically, the dipeptide will be cyclized byrefluxing it for about 8-24 hours, preferably about 18 hours. Finally,the protecting group is removed from the diketopiperazine. In doing so,the use of strong acids (mineral acids, such as sulfuric or hydrochloricacids), strong bases (alkaline bases, such as potassium hydroxide orsodium hydroxide), and strong reducing agents (e.g., lithium aluminumhydride) should be avoided, in order to maintain the chirality of thefinal compound.

Dipeptides made on solid phase resins can be cyclized and released fromthe resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance,the resin having an N-alkylated dipeptide attached is suspended intoluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) ortriethylamine (e.g., 4%). Typically, basic cyclization conditions arepreferred for their faster cyclization times.

Other methods of cyclizing dipeptides and of making diketopiperazinesare known in the art and can be used in the preparation ofdiketopiperazines useful in the practice of the invention. See, e.g.,those references listed above.

To prepare the diketopiperazine of formula I wherein the amino acid sidechains are derivatized, amino acid derivatives can be used in thesynthesis of the dipeptides, the dipeptides can be derivatized and/orthe diketopiperazines can be derivatized, as is known in the art. See,e.g., those references cited above. Also see U.S. Pat. No. 5,589,501,U.S. Patent Appl. Pub. No. 2005/0215468, EP Patent Application No.1,445,323, Chang et al., J. Med. Chem., 16(11):1277-1280 (1973). Thecomplete disclosures of these references are incorporated herein byreference, as exemplary methods of making amino acid derivatives with R²side chains (e.g., aspartic acid and glutamic acid derivatives).

The diketopiperazines of formula I include all possible stereoisomersthat can be obtained by varying the configuration of the individualchiral centers, axes or surfaces. In other words, the diketopiperazinesof formula I include all possible diastereomers, as well as all opticalisomers (enantiomers).

When a diketopiperazine of formula I contains one or more chiralcenters, the compound can be synthesized enantioselectively or a mixtureof enantiomers and/or diastereomers can be prepared and separated. Theresolution of the compounds of the present invention, their startingmaterials and/or the intermediates may be carried out by knownprocedures, e.g., as described in the four volume compendium OpticalResolution Procedures for Chemical Compounds: Optical ResolutionInformation Center, Manhattan College, Riverdale, N.Y., and inEnantiomers, Racemates and Resolutions, Jean Jacques, Andre Collet andSamuel H. Wilen; John Wiley & Sons, Inc., New York, 1981, which areincorporated herein in their entirety. Basically, the resolution of thecompounds is based on the differences in the physical properties ofdiastereomers by attachment, either chemically or enzymatically, of anenantiomerically pure moiety, resulting in forms that are separable byfractional crystallization, distillation or chromatography.

The pharmaceutically-acceptable salts of the diketopiperazines offormula I may also be used in the practice of the invention.Pharmaceutically-acceptable salts include conventional non-toxic salts,such as salts derived from inorganic acids (such as hydrochloric,hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids(such as acetic, propionic, succinic, glycolic, stearic, lactic, malic,tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic,ascorbic acid, and the like) or bases (such as the hydroxide, carbonateor bicarbonate of a pharmaceutically-acceptable metal cation or organiccations derived from N,N-dibenzylethylenediamine, D-glucosamine, orethylenediamine). The salts are prepared in a conventional manner, e.g.,by neutralizing the free base form of the compound with an acid.

“Prodrug” means any compound which releases an active parent drugaccording to formula I in vivo when such prodrug is administered to ananimal. Prodrugs of a diketopiperazine of formula I are prepared bymodifying one or more functional group(s) present in thediketopiperazine of formula I in such a way that the modification(s) maybe cleaved in vivo to release the parent drug (i.e., thediketopiperazine of formula I). Prodrugs include diketopiperazines offormula I wherein a hydroxy, amino, or sulfhydryl group in a compound offormula I is bonded to any group that may be cleaved in vivo to generatethe free hydroxyl, amino, or sulfhydryl group, respectively. Examples ofprodrugs include, but are not limited to, esters (e.g., acetate,formate, and benzoate derivatives), carbamates (e.g.,N,N-dimethylaminocarbonyl) of hydroxy functional groups in compounds offormula I, and the like.

As noted above, the invention provides a method of treating aproliferative disease or condition. A proliferative disease or conditionis a disease or condition causing, caused by, involving, or exacerbatedby, proliferation of cells. Specific proliferative diseases andconditions that can be treated with a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof, include bloodvessel proliferative disorders, cancer, mesangial cell proliferationdisorders, fibrotic disorders and hyperproliferative skin disorders.

Blood vessel proliferative disorders include angiogenic diseases andconditions. An angiogenic disease or condition is a disease or conditioncausing, caused by, involving, exacerbated by, or dependent onangiogenesis. Angiogenesis is the process of new blood vessel formationin the body. Angiogenesis is also used herein to mean the same as, or toinclude, neovascularization, vascularization, arterialization andvasculogenesis.

A diketopiperazine of the invention or a pharmaceutically-acceptablesalt or prodrug thereof will inhibit angiogenesis and can be used totreat an angiogenic disease or condition. Specific angiogenic diseasesand conditions treatable according to the invention include neoplasticdiseases (e.g., tumors (e.g., tumors of the bladder, brain, breast,cervix, colon, rectum, kidney, lung, ovary, pancreas, prostate, stomachand uterus) and tumor metastasis), benign tumors (e.g., hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyrogenic granulomas),hypertrophy (e.g., cardiac hypertrophy induced by thyroid hormone),connective tissue disorders (e.g., arthritis and atherosclerosis),psoriasis, ocular angiogenic diseases (e.g., diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, and rubeosis),cardiovascular diseases, cerebral vascular diseases, endometriosis,polyposis, obesity, diabetes-associated diseases, hemophiliac joints,inflammation and autoimmunity. The diketopiperazines of the inventionwill be particularly useful for the treatment of neoplastic diseases andocular angiogenic diseases (especially diabetic retinopathy and maculardegeneration). The diketopiperazines of the invention can also be usedto inhibit the vascularization required for embryo implantation, therebyproviding a method of birth control.

The invention also provides a method of treating a cancer or aprecancerous condition. Cancers treatable with a diketopiperazine offormula I, or a pharmaceutically-acceptable salt or prodrug thereof,include carcinomas, sarcomas, osteoscarcomas, lymphomas, leukemias,hematologic malignancies, cancer syndromes, malignant tumors, andmetastases. Specific cancers treatable according to the inventioninclude brain cancers, head and neck cancers, breast cancers, cardiaccancers, ovarian cancers, cervical cancers, endometrial cancers,urogenital cancers, prostate cancers, gastric cancers, colorectalcancers, pancreatic cancers, bladder cancers, thyroid cancers, hepaticcancers, lung cancers, bone cancers, skin cancers and Kaposi's sarcomas.Specific cancer syndromes treatable according to the invention includeBannayan-Zonana syndrome, Cowden disease and Lhermitte-Duclos disease.Specific malignant tumors treatable according to the invention includemalignant tumors of the bladder, bone, brain, breast, cervix, colon,heart, kidney, liver, lung, lymph tissue, ovary, pancreas, prostate,rectum, skin, stomach, thyroid, urogenital and uterus.

The diketopiperazines of the invention are especially useful for thetreatment of breast cancer and melanoma and for the treatment ofmetastases. The diketopiperazines of the invention are also especiallyuseful for the treatment of malignant brain tumors, including primarytumors and metastatic (secondary) tumors. About half of all primarybrain tumors are gliomas. Gliomas include astrocytomas (e.g., pilocyticastrocytomas, low-grade astrocytomas, anaplastic (high-grade)astrocytomas and glioblastomas multiforme), brain stem gliomasependymomas, ganglioneuromas, juvenile pilocytic gliomas, mixed gliomas,oligodendrogliomas and optic nerve gliomas. Glioblastomas are the mostcommon malignant brain tumors in adults and are probably the mostresistant of all cancers to treatment. Other primary brain tumorsinclude carniopharyngiomas, medulloblastomas, pineal tumors, pituitaryadenomas, primitive neuroectodermal tumors and vascular tumors.Metastatic brain tumors are tumors that have spread to the brain fromanother part of the body. The most common cancers that metastasize tothe brain include breast, melanoma and lung cancers. Metastatic braintumors are the most common form of brain tumor and considerablyoutnumber primary brain tumors.

Precancerous conditions treatable with a diketopiperazine of formula I,or a pharmaceutically-acceptable salt or prodrug thereof, includemyelodysplastic syndrome, aplastic anemia, cervical lesions, skin nevi(pre-melanoma), prostatic intraepithelial (intraductal) neoplasia,ductal carcinoma in situ, Helicobacter pylori infections of the stomach,colon polyps, severe hepatitis or cirrhosis (especially virally-inducedhepatitis) of the liver, and other premalignant conditions that canprogress to cancer.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial cellproliferative disorders include renal diseases, such asglomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic microangiopathy syndromes and glomerulopathies.

Fibrotic disorders are diseases or conditions causing, caused by,involving or exacerbated by the abnormal formation of extracellularmatrices, unwanted or excessive fibrosis, or both. Fibrotic disorderscan occur in, for instance, skin, liver, kidney, heart or lung tissue.Fibrotic disorders include scarring (e.g., keloid formation andhypertrophic scars), scleroderma, kidney fibrosis (e.g., glomerularsclerosis or renal tubulointerstitial fibrosis), pulmonary fibrosis(including idiopathic pulmonary fibrosis), cardiac fibrosis,chemotherapy/radiation-induced lung fibrosis, pancreatitis,atherosclerosis, restenosis, inflammatory bowel disease, Crohn'sdisease, arthritis, cancer (e.g., invasive breast cancer, stromal richmammary tumors, dermatofibromas, angiolipoma and angioleiomyoma),fascitis, general fibrosis syndrome (characterized by replacement ofnormal muscle tissue by fibrous tissue in varying degrees), liverfibrosis (e.g., hepatic cirrhosis), acute fibrosis (e.g., in response tovarious forms of trauma, including accidental injuries, infections,surgery, burns, radiation or chemotherapy treatment), maculardegeneration and diabetic retinopathy.

Hyperproliferative skin disorders include psoriasis, skin cancer andepidermal hyperproliferation.

The diketopiperazines of the invention, or a pharmaceutically-acceptablesalt or prodrug thereof, can also be used to treat viral infections.Specific viral infections treatable according to the invention includeinfections caused by hepatitis B virus, hepatitis C virus, rubellavirus, human immunodeficiency virus (HIV), human herpesvirus 4(Epstein-Barr virus), human herpesvirus 5 (human cytomegalovirus orHCMV), human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus orKSHV), human papillomarvirus (HPV), polyomaviruses, human respiratorysyncytial virus (RSV), adenovirus and influenza virus.

The invention also provides a method of inhibiting the production,release or both of matrix metalloproteinase-9 (MMP-9) by cells. Themethod comprises contacting the cells with an effective amount of adiketopiperazine of formula I or a pharmaceutically-acceptable salt orprodrug thereof. The cells can be contacted with the diketopiperazine byany method known in the art. In particular, the cells can be contactedwith the diketopiperazine in vivo by administering an effective amountof the diketopiperazine to an animal.

Cells that produce and/or release MMP-9 include those cells found in oraround a tumor. Such cells include the tumor cells themselves, stromalcells, eosinophils, macrophages, neutrophils and endothelial cells. SeeThiennu H. Vu and Zena Werb, “Gelatinase B: Structure, Regulation, andFunction,” pages 115-148, in Matrix Metalloproteinases (Academic Press,Editors William C. Parks and Robert P. Mecham, 1998). Cells that produceand/or release MMP-9 also include cells involved in inflammation. See,e.g, Id., Solakivi et al., Lipids in Health and Disease, 8(11) (EpubMar. 30, 2009), Lu et al., J. Leukocyte Biol., 78:259-265 (2005) andAmin et al., Genes Cells, 8:515-523 (2003).

The invention also provides a method of inhibiting the activation of Aktby cells. The method comprises contacting the cells with an effectiveamount of a diketopiperazine of formula I or apharmaceutically-acceptable salt or prodrug thereof. The cells can becontacted with the diketopiperazine by any method known in the art. Inparticular, the cells can be contacted with the diketopiperazine in vivoby administering an effective amount of the diketopiperazine to ananimal.

Protein kinases are involved in the signal transduction pathways linkinggrowth factors, hormones and other cell regulation molecules to cellgrowth, survival and metabolism. One such protein kinase is Akt kinase.Akt kinase, also known as protein kinase B, is a serine/threonine kinasethat plays a central role in promoting the proliferation and survival ofa wide range of cell types, thereby protecting cells from apoptosis. Anumber of protein kinases and phosphatases regulate the activity of Akt.For instance, activation of Akt is mediated by phosphatidylinositol3′-OH kinase (PI3 kinase or PI3K). Activated PI3K producesphosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P₃) at the inner sideof the plasma membrane. The increase in PI(3,4,5)P₃ recruits Akt to theinner membrane, where it is activated. Akt can also be activated bygrowth signals that are independent of PI3K. Full activation of Aktrequires phosphorylation at two sites by two different kinases.

Activated Akt modulates the function of numerous substrates involved inthe regulation of cell proliferation, growth and survival, and of cellcycle entry and progression, and activated Akt is involved in theregulation of numerous cellular processes, including transcription,differentiation, metabolism, apoptosis, migration, metastasis,angiogenesis and fibrosis. As a consequence, Akt plays a role innumerous diseases and conditions.

In particular, Akt is known to play a critical role in cancer.Activation of Akt contributes to tumorigenesis in many types of tissues,including breast, ovarian, brain, prostate, skin and lymph tissues.Elevated levels of activated Akt have been detected in a variety ofcancers, including ovarian, breast, prostate, pancreatic, gastric,colorectal, brain, thyroid, lung, skin, leukemia and undifferentiatedtumors (suggesting that Akt may be associated with tumor aggressivenessand progression). In addition, it has been found that Akt isconstitutively active in a wide array of cancers. The phosphatase PTENis a critical negative regulator of Akt, and its function is lost inmany cancers, including breast and prostate carcinomas, glioblastomasand several cancer syndromes, including Bannayan-Zonana syndrome, Cowdendisease and Lhermitte-Duclos disease. Tumor cells without functionalPTEN show elevated levels of activated Akt. Cancer treatment bychemotherapy and gamma-irradiation kills target cells primarily byinduction of apoptosis, and the anti-apoptotic effects of activated Aktcontribute to both chemotherapeutic resistance and radiation resistance.Activated Akt also contributes to tumor invasiveness and metastasis. Aktactivation is associated with increased expression and secretion ofmatrix metalloproteases MMP-9 and MMP-2, and Akt has the ability toup-regulate angiogenesis, both of which also contribute to tumorsurvival.

Akt also plays a role in the life cycle of viruses. See, e.g., Cooray,J. Gen. Virol., 85:1065-1076 (2004) and PCT application WO 2007/149730.In particular, inhibition of apoptosis has become recognized as animportant contributory factor in virus survival. Apoptotic inhibitioncontributes to the establishment of latent and chronic infections andhas been implicated in viral oncogenesis. Virus modulation of thePI3K/Akt pathway provides an alternative to the expression of viraloncogenes or the direct inhibition of pro-apoptotic proteins. It hasbecome evident that many viruses require upregulation of this pathway tosustain long-term infections and it is modulated, in some cases, byspecific viral products to create an environment favorable for cellulartransformation. In other cases, PI3K/Akt signaling simply helps tocreate an environment favorable for virus replication and virionassembly.

Akt plays a role in angiogenesis and fibrosis. Angiogenesis and fibrosisare key components in development, growth, wound healing andregeneration. Activation of Akt is sufficient to induce angiogenesis,and activated Akt plays an important role in several of the processesinvolved in angiogenesis. See, e.g., Jiang and Liu, Current Cancer DrugTargets, 8:19-26 (2008); Sheng et al., J. Cell. Physiol., 218:451-454(2009). Activation of the Akt pathway results in the production ofconnective tissue growth factor (CTGF). CTGF is a potent growth factorthat has been shown to play a role in fibroblast proliferation, celladhesion and the stimulation of extracellular matrix (ECM) production,and CTGF is a potent activator of fibrosis. Accordingly, Akt is a targetof choice for anti-angiogenesis therapy for cancer and other angiogenicdiseases and conditions and for treating fibrotic disorders.

Due to its pivotal role in controlling cell proliferation, apopotosisand cell migration, Akt is the master regulator of theproliferative/migratory response of vessel wall cells to injury. PCTapplication WO 03/032809. Accordingly, restenosis of vessels afterangioplasty and narrowing of implanted blood vessels (such as arteries,veins, vascular grafts and conduits) following implantation can beprevented or reduced by inhibiting Akt activity in the cells of thevessel. Id. The Akt inhibitor is preferably administered locally to theblood vessel (such as through a catheter or by being provided as part ofa coating on a stent). Id.

Akt activation has also been reported to play a role in inflammation andautoimmunity. See, e.g, Ottonello et al., Br. J. Pharmacol., (Mar. 25,2009) (Epub ahead of print) (PMID 19338579), Solakivi et al., Lipids inHealth and Disease, 8(11) (Epub Mar. 30, 2009), Rane et al., Front.Biosci., 14:2400-2412 (2009), Baker et al., J. Immunol.,182(6):3819-3826 (2009), Takeshima et al., BMC Microbiol., 9:36 (2009),Patel et al., Immunol. Res., 31(1):47-55 (2005), Lu et al., J. LeukocyteBiol., 78:259-265 (2005), and Amin et al., Genes Cells, 8:515-523(2003).

For general background on Akt and its involvement in various diseasesand conditions, see, e.g., U.S. Pat. Nos. 7,175,844, 7,220,539 and7,378,403, U.S. Pub. Patent Appl. No., 2008/0009507, PCT applicationsnumbers WO 2009/032651, WO 2009/009793, WO 2007/149730, WO 2004/086038and WO 03/032809, Russo et al., Int. J. Oncol., 34(6):1481-1489 (June2009) (abstract, PMID 19424565, full article in process), Sheng et al.,J. Cell. Physiol., 218:451-454 (2009), Ottonello et al., Br. J.Pharmacol., (Mar. 25, 2009) (Epub ahead of print) (PMID 19338579),Solakivi et al., Lipids in Health and Disease, 8(11) (Epub Mar. 30,2009), Rane et al., Front. Biosci., 14:2400-2412 (2009), Baker et al.,J. Immunol., 182(6):3819-3826 (2009), Takeshima et al., BMC Microbiol.,9:36 (2009), Sinnberg et al., J. Invest. Dermatol., PMID 19078992,abstract of article Epub Dec. 11, 2008, Cho and Park, Int. J. Mol. Sci.,9(11):2217-2230 (November 2008), Qiao et al., Cell Cycle,7(19):2991-2996 (Epub Oct. 13, 2008), Jiang and Liu, Current Cancer DrugTargets, 8:19-26 (2008), Dida et al., Experimental Hematology,36:1343-1353 (2008), Ji et al., Recent Pat. Biotechnol., 2(3):218-226(2008), Wang et al., J. Neuroscience, 26(22):5996-6003 (2006), Chen etal., Current Medicinal Chemistry—Anti-Cancer Agents, 9(6):575-589(November 2005), Kim et al., Endocrinology, 146(10):4456-4463 (2005),Patel et al., Immunol. Res., 31(1):47-55 (2005), Lu et al., J. LeukocyteBiol., 78:259-265 (2005), Cooray, J. Gen. Virol., 85:1065-1076 (2004),and Amin et al., Genes Cells, 8:515-523 (2003), the complete disclosuresof all of which are incorporated herein by reference.

The diketopiperazines of the present invention, or apharmaceutically-acceptable salt or prodrug thereof, can be used totreat an Akt-mediated disease or condition. An Akt-mediated disease orcondition is a disease or condition causing, caused by, involving, orexacerbated by, activation of Akt. Akt-mediated diseases and conditionsinclude proliferative diseases and conditions, angiogenic diseases andconditions, cancer, fibrotic disorders, restenosis, viral infections,inflammation and autoimmunity.

While not wishing to be bound by any theory, it is presently believedthat it is the R² side chain of the diketopiperazines of formula I thatis primarily responsible for their activity. Further, it is presentlybelieved that optimum activity is obtained when R² has formula II, R⁵ isaryl, heteroaryl or aryloxy, n is 1 and R⁶ is methyl. Preferably, R⁵ isaryl or aryloxy. More preferably, R⁵ is phenyl or phenoxy, mostpreferably phenyl. Also, preferably, R⁵ is in the 4 (para) position onthe ring.

It is to be understood that the scope of this invention encompasses notonly the use of the diketopiperazines of formula I themselves, but alsothe pharmaceutically-acceptable salts and prodrugs thereof. In addition,the present invention contemplates the use of the isomers of thediketopiperazines of formula I, and of the pharmaceutically-acceptablesalts and prodrugs thereof, including pure isomers and various mixturesof isomers.

“Inhibit” or “inhibiting” is used herein to mean to reduce (wholly orpartially) or to prevent.

“Treat,” “treating” or “treatment” is used herein to mean to reduce(wholly or partially) the symptoms, duration or severity of a disease orcondition, including curing the disease, or to prevent, or reduce theincidence of, the disease or condition (i.e., to cause the symptoms ofthe disease or condition not to develop in an animal that may be exposedor predisposed to the disease or condition, but does not yet experienceor display symptoms of the disease or condition).

By “effective amount” is meant the amount of a compound that, whenadministered to an animal for treating a disease or condition or forcausing an effect is sufficient to do so. The “effective amount” can andwill most likely vary depending on the compound, the disease orcondition and its severity, or the effect sought to be caused, and theage, weight, etc., of the animal to be treated (see below).

To treat a animal, a diketopiperazine of formula I, or apharmaceutically-acceptable salt or prodrug thereof, is administered tothe animal. Preferably, the animal is a mammal, such as a rabbit, goat,dog, cat, horse or human. Effective dosage forms, modes ofadministration and dosage amounts for the compounds of the invention maybe determined empirically, and making such determinations is within theskill of the art. It is understood by those skilled in the art that thedosage amount will vary with the particular compound(s) employed, thedisease or condition to be treated, the severity of the disease orcondition, the route(s) of administration, the rate of excretion of thecompound, the duration of the treatment, the identity of any other drugsbeing administered to the animal, the age, size and species of theanimal, and like factors known in the medical and veterinary arts. Ingeneral, a suitable daily dose of a compound of the present inventionwill be that amount of the compound which is the lowest dose effectiveto produce a therapeutic effect. However, the daily dosage will bedetermined by an attending physician or veterinarian within the scope ofsound medical judgment. If desired, the effective daily dose may beadministered as two, three, four, five, six or more sub-doses,administered separately at appropriate intervals throughout the day.Administration of the compound should be continued until an acceptableresponse is achieved.

The compounds of the present invention (i.e., diketopiperazines offormula I and pharmaceutically-acceptable salts and prodrugs thereof)may be administered to an animal patient for therapy by any suitableroute of administration, including orally, nasally, rectally, vaginally,parenterally (e.g., intravenously, intraspinally, intraperitoneally,subcutaneously, or intramuscularly), intracisternally, transdermally,intracranially, intracerebrally, and topically (including buccally andsublingually). The preferred routes of administration are orally andtopically.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The pharmaceuticalcompositions of the invention comprise a compound or compounds of theinvention as the active ingredient(s) in admixture with one or morepharmaceutically-acceptable carriers and, optionally, with one or moreother drugs. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the animal. Pharmaceutically-acceptable carriers are wellknown in the art. Regardless of the route of administration selected,the compounds of the present invention are formulated intopharmaceutically-acceptable dosage forms by conventional methods knownto those of skill in the art. See, e.g., Remington's PharmaceuticalSciences.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient (i.e., one or more diketopiperazines of formula Iand/or pharmaceutically-acceptable salts and/or prodrugs thereof) ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monosterate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugars, as well ashigh molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat releases the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active ingredient, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of compoundsof the invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, drops and inhalants. The activeingredient may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to theactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active ingredient,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of compounds of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating one ormore compounds of the invention in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical formulations include those suitable for administration byinhalation or insufflation or for nasal or intraocular administration.For administration to the upper (nasal) or lower respiratory tract byinhalation, the compounds of the invention are conveniently deliveredfrom an insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof one or more compounds of the invention and a suitable powder base,such as lactose or starch. The powder composition may be presented inunit dosage form in, for example, capsules or cartridges, or, e.g.,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator, insufflator or a metered-dose inhaler.

For intranasal administration, compounds of the invention may beadministered by means of nose drops or a liquid spray, such as by meansof a plastic bottle atomizer or metered-dose inhaler. Liquid sprays areconveniently delivered from pressurized packs. Typical of atomizers arethe Mistometer (Wintrop) and Medihaler (Riker).

Drops, such as eye drops or nose drops, may be formulated with anaqueous or nonaqueous base also comprising one or more dispersingagents, solubilizing agents or suspending agents. Drops can be deliveredby means of a simple eye dropper-capped bottle or by means of a plasticbottle adapted to deliver liquid contents dropwise by means of aspecially shaped closure.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The diketopiperazines of formula I, or a pharmaceutically-acceptablesalt or prodrug thereof, may be given alone to treat a disease orcondition according to the invention. Alternatively, thediketopiperazines of formula I, or a pharmaceutically-acceptable salt orprodrug thereof, may be given in combination with one or more othertreatments or drugs suitable for treating the disease or condition. Thecompounds of the present invention can be administered prior to, inconjunction with (including simultaneously with) or after, the othertreatment or drug.

For instance, when used to treat cancer, the compounds of the presentinvention can be administered prior to, in conjunction with or after,another anti-cancer treatment. Such anti-cancer treatments includesurgery, radiation or chemotherapy using any of a variety of anti-canceragents. Typically, any anti-cancer agent that has activity versus asusceptible tumor being treated may be administered prior to, inconjunction with or after, a diketopiperazine, or apharmaceutically-acceptable salt or prodrug thereof, in the treatment ofcancer according to the present invention. Examples of such agents canbe found in Devita and Hellman (editors), Cancer Principles and Practiceof Oncology, 6^(th) edition, Feb. 15, 2001, Lippincott Williams &Wilkins (publishers), U.S. Pub. Patent Appl. No. 2008/0009507, PCTApplication No. WO 2009/009793 and PCT Application No. WO 2009/032651,the complete disclosures of all of which are incorporated herein byreference as disclosing exemplary anti-cancer agents to be administeredwith diketopiperazine in methods of the present invention. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Typical anti-cancer agents useful in thepresent invention include anti-microtubule agents, anti-mitotic agents,platinum coordination complexes, alkylating agents, antibiotic agents,antimetabolites, hormones and hormonal analogs, topoisomerase Iinhibitors, topoisomerase II inhibitors, angiogenesis inhibitors, signaltransduction pathway inhibitors, proapoptotic agents andimmunotherapeutic agents.

Anti-microtubule or anti-mitotic agents are phase specific agents activeagainst the microtubules of tumor cells during M or the mitosis phase ofthe cell cycle. Examples of such agents include diterpenoids (e.g.,paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine,vincristine and vinorelbine).

Platinum coordination complexes are non-phase specific anti-canceragents which are interactive with DNA. The platinum complexes entertumor cells, undergo aquation and form intra- and interstrand crosslinkswith DNA causing adverse biological effects to the tumor. Examples ofplatinum coordination complexes include cisplatin and carboplatin.

Alkylating agents are non-phase anti-cancer specific agents and strongelectrophiles. Typically, alkylating agents form covalent linkages, byalkylation, to DNA through nucleophilic moieties of the DNA moleculessuch as phosphate, amino, sulfhydryl, hydroxyl, carboxyl and imidazolegroups. Such alkylation disrupts nucleic acid function leading to celldeath. Examples of alkylating agents include nitrogen mustards (e.g.,cyclophosphamide, melphalan and chlorambucil), alkyl sulfonates (e.g,busulfan), nitrosoureas (e.g, carmustine), triazenes (e.g., dacarbazine)and imidazotetrazines (e.g., temozolomide).

Antibiotic anti-cancer agents are non-phase specific agents which bindor intercalate with DNA. Typically, such action results in stable DNAcomplexes or strand breakage, which disrupts ordinary function of thenucleic acids leading to cell death. Examples of antibiotic anti-canceragents include actinomycins (e.g., dactinomycin), anthrocyclins (e.g.,daunorubicin and doxorubicin) and bleomycins.

Topoisomerase I inhibitors include camptothecin and camptothecinderivatives (e.g, irinotecan, topotecan and the various optical forms of7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptothecin).Camptothecins' cytotoxic activity is believed to be related to itstopoisomerase I inhibitor activity.

Topoisomerase II inhibitors include epipodophyllotoxins (e.g., etoposideand teniposide). Epipodophyllotoxins are phase specific anti-canceragents derived from the mandrake plant. Epipodophyllotoxins typicallyaffect cells in the S and G₂ phases of the cell cycle by forming aternary complex with topoisomerase II and DNA causing DNA strand breaks.The strand breaks accumulate and cell death follows.

Antimetabolite anti-cancer agents are phase specific agents that act atS phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis orby inhibiting purine or pyrimidine base synthesis and thereby limitingDNA synthesis. Consequently, S phase does not proceed and cell deathfollows. Examples of antimetabolite anti-cancer agents includefluorouracil, methotrexate, cytarabine, mercaptopurine, thioguanine,hydroxyurea and gemcitabine.

Hormones or hormonal analogs are useful compounds for treating cancersin which there is a relationship between the hormone(s) and growth orlack of growth of the cancer. Examples of hormones and hormonal analogsuseful in cancer treatment include adrenocorticosteroids (e.g.,prednisone and prednisolone), aminoglutethimide and other aromataseinhibitors (e.g., anastrozole, letrazole, vorazole and exemestane),progestins (e.g., megestrol acetate), estrogens, androgens andanti-androgens (e.g., flutamide, nilutamide, bicalutamide, cyproteroneacetate and 5α-reductases, such as finasteride and dutasteride),anti-estrogens (e.g., tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene and selective estrogen receptor modulators),gonadotropin-releasing hormone and analogs thereof which stimulate therelease of leutinizing hormone and/or follicle stimulating hormone(e.g., goserelin aceteate and luprolide).

Angiogenesis inhibitors include anti-VEGF antibodies, inhibitors ofintegrin alpha_(v)beta₃, endostatin, angiostatin, danazol and thosemethylphenidate derivatives described in U.S. Pub. Patent Appl. No.20060189655, particularly α-[phenyl-4-phenyl]-2-piperidineacetic acidmethyl ester and α-[phenyl-4-phenoxy]-2-piperidineacetic acid methylester (i.e., those derivatives of methylphenidate(α-phenyl-2-piperidineacetic acid methyl ester) having the hydrogen ofthe phenyl group at the 4 (para) position replaced with phenyl orphenoxy).

Signal transduction pathway inhibitors are those inhibitors which blockor inhibit a chemical process which evokes an intracellular change,particularly cell proliferation or differentiation. Signal transductioninhibitors useful in the present invention include inhibitors ofreceptor tyrosine kinases, non-receptor tyrosine kinases,serine/threonine kinases, PI3Ks, myoinositol signaling and Rasoncogenes, and SH2/SH3 domain blockers. Suitable inhibitors aredescribed in PCT Application No. WO 2009/032651 and the references citedtherein, and include kinase inhibitors, receptor antagonists,antibodies, ribozymes and anti-sense oligonucleotides. Specific examplesincludeN-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}6-[5-({[2-(methanesulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine(also known as lapatanib and Tykerb), inhibitors of farnesyltransferase,geranyl-geranyl transferase and CAAX proteases, Imclone C225 EGFRspecific antibody, Herceptin (trastuzamab), erbB2 antibody, 2CB VEGFR2specific antibody, BAY-43-9006, CI-1040, PD-098059, Wyeth CCI 779, andLY294002 and cell cycle signaling inhibitors (e.g., inhibitors ofcyclin-dependent kinases).

Proapoptotic agents include Genta's G3139 bcl-2 antisenseoligonucleotide which downregulates the expression of bcl-2 in tumorsand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Immunotherapeutic agents include Imclone C225 EGFR specific antibody,Herceptin (trastuzamab), alemtuzumab, Erbitux (cetuximab), Avastin(bevacizumab), gemtuzumab, iodine 131 tositumomab, rituximab, erbB2antibody, 2CB VEGFR2 specific antibody, anti-VEGF antibodies, andvaccines.

When used to treat a viral infection, the compounds of the presentinvention can be administered prior to, in conjunction with or after,another anti-viral drug. Anti-viral drugs include amantadine,rimantadine, pleconaril, lamivudine, fomivirsen, rifampicin, zidovudine,Relenza (zanamivir), Tamiflu (oseltamivir phosphate), Zovirax(acyclovir), interferons and antibodies.

The following Examples are intended to illustrate embodiments of theinvention and are not intended to limit the invention.

EXAMPLES Example 1 Synthesis ofbiphenyl-4-yl-(3,6-dioxo-piperazin-2-yl)-acetic acid methyl ester (Cpd.5)

Biphenyl-4-yl-(3,6-dioxo-piperazin-2-yl)-acetic acid methyl ester (Cpd.5) was synthesized as described below and illustrated in FIG. 1.

(A) Synthesis of biphenyl-4-yl-acetic acid methyl ester (Cpd. 1)

To a stirred solution of 4-biphenylacetic acid (from Aldrich, 196487) (2g., 9.4 mmol) in methanol (50 mL) at 0° C. was added drop-wise,thionylchloride (from Aldrich, 230464) (4.1 mL, 56.5 mmol), and thereaction was stirred for 0.5 hours at room temperature. The reactionmixture was concentrated in vacuo and the residue partitioned betweenethylacetate (100 mL) and water (100 mL). The organic extract was washedwith sodium bicarbonate (saturated solution, 100 mL), brine (saturatedsolution, 100 mL) and dried over magnesium sulphate. The solvents wereremoved in vacuo to afford Cpd. 1 as a yellow oil (2.1 g, ˜100% yield).¹HNMR (CDCl₃) δH, 3.67, 2H (s, CH₂), 3.71, 3H (s, CH₃), 7.31-7.37, 3H(m, Ar—H), 7.40-7.45, 2H (m, Ar—H), 7.52-7.59, 4H (m, Ar—H); LCMSanalysis (solvent MeCN/H₂O/0.1% HCO₂H, 5-95% gradient/H₂O 2.5 min, 95%1.5 min., Phenomenex C18 reverse phase, flow rate 1 mL/min.) HPLCretention time 2.39 min.; mass found 167 (M-CO₂Me).

(B) Synthesis of biphenyl-4-yl-bromo-acetic acid methyl ester (Cpd. 2)

To a stirred solution of biphenyl-4-yl-acetic acid methyl ester (5 g.,22.1 mmol) in carbon tetrachloride (from Acros, 16772) (25 mL) was addedN-bromosuccinimide (from Aldrich, 18350) (3.9 g., 22.1 mmol) and benzoylperoxide (from Acros, 21178) (0.54 g, 2.2 mmol) and the reaction washeated to reflux for 48 hours. The reaction mixture was cooled to roomtemperature, filtered and the filtrate concentrated in vacuo to affordCpd. 2 as a crude yellow oil (6.24 g., 93% yield). ¹HNMR (CDCl₃) δH,3.81, 3H (s, CH₃), 5.41, 1H (s, Br—CH), 7.33-7.39, 2H (m, Ar—H),7.41-7.46, 3H (m, Ar—H), 7.54-7.63, 4H (m, Ar—H); LCMS analysis (solventMeCN/H₂O/0.1% HCO₂H, 5-95% gradient/H₂O 2.5 min, 95% 4 min., PhenomenexC18 reverse phase, flow rate 1 mL/min.) HPLC retention time 4.39 min.

(C) Synthesis of 2-amino-3-biphenyl-4-yl-succinic acid (Cpd. 3)

To a stirred solution of diethylacetamido malonate (from Fisher, 11393)(0.53 mg., 12.5 mmol) in ethanol (10 mL) at 0° C., was added drop-wiseto a solution of sodium ethoxide (from Fluka, 71212) (0.7 mL, 2%solution in ethanol) and the reaction was stirred at room temperaturefor 0.5 hours. A solution of biphenyl-4-yl-bromo-acetic acid methylester (0.5 g, 1.6 mmol) in ethanol (5 mL) was added drop-wise and thereaction stirred at room temperature for a further 1.5 hours. Thereaction was quenched by the addition of water (25 mL) and extractedwith ethylacetate (3×50 mL). The organic extracts were combined, driedover magnesium sulphate and concentrated in vacuo to afford a cruderesidue. The crude residue was purified using flash chromatography(SiO₂) eluting with ethylacetate and isohexane (1:1) to afford2-acetylamino-3-biphenyl-4-yl-2-ethyoxycarbonyl-succinic acid 1-ethylester 4-ethyl ester as a pale yellow solid (0.49 g, 68% yield) (contains˜10% of the corresponding 4-methyl ester). ¹HNMR (CDCl₃) δH (ppm); 1.2,3H (t, CH₃ J=7.02 Hz), 1.22, 3H (t, CH₃ J=7.02 Hz), 1.3, 3H (t, CH₃J=7.02 Hz), 1.9, 3H (s, CH₃), 4.04-4.23, 4H (m, 2×CH₂), 4.31, 2H (q, CH₂J=7.02 & 14.3 Hz), 5.01, 1H (s, CH), 6.5, 1H (br s, NH), 7.31-7.36, 1H(m, Ar—H), 7.40-7.46, 4H (m, Ar—H), 7.5-7.54, 2H (m, Ar—H), 7.56-7.60,2H (m, Ar—H); LCMS analysis (solvent MeCN/H₂O/0.1% HCO₂H, 5-95%gradient/H₂O 2.5 min, 95% 4 min., Phenomenex C18 reverse phase, flowrate 1 mL/min.) HPLC retention times 4.68 and 4.9 min.(diastereoisomers); mass found 465 (M+H).

A solution of 2-acetylamino-3-biphenyl-4-yl-2-ethoxycarbonyl-succinicacid 1-ethyl ester 4-ethyl ester (0.33 g., 0.76 mmol) in 9N hydrochloricacid (10 mL) was heated at reflux for 7 hours. The reaction mixture wascooled to room temperature and concentrated in vacuo to afford Cpd. 3 asa white crystalline solid (0.21 g., 96% yield). ¹HNMR (DMSO-d₆) δH(ppm); 4.24, 1H (d, CH J=7.02 Hz), 4.38, 0.5H (d, CH J=7.02 Hz), 4.44,0.5H (d, CH J =7.02 Hz), 7.33-7.39, 2H (m, Ar—H), 7.40-7.48, 3H (m,Ar—H), 7.62-7.69, 4H (m, Ar—H), 8.4, 3H (br s, NH₃ ⁺)(diastereoisomers); LCMS analysis (solvent MeCN/H₂O/0.1% HCO₂H, 5-95%gradient/H₂O 2.5 min, 95% 4 min., Phenomenex C18 reverse phase, flowrate 1 mL/min.) HPLC retention time 5.9 min.; mass found 286 (M+H).

(D) Synthesis of 2-amino-3-biphenyl-4-yl-succinic acid dimethyl esterhydrochloride (Cpd. 4)

To a stirred solution of 2-amino-3-biphenyl-4-yl-succinic acidhydrochloride (1.54 g., 48 mmol) in methanol (100 mL) at 0° C. was addeddropwise to thionyl chloride (4.2 mL, 57.5 mmol) and the reaction washeated at 40° C. for 29 hours. The reaction mixture was concentrated invacuo and the residue partitioned between saturated sodium bicarbonate(100 mL) and ethylacetate (100 mL). The aqueous layer was washed withethyl acetate (2×50 mL) and the organics combined, dried over magnesiumsulphate and concentrated in vacuo to afford the free base as a paleyellow oil. The residue was dissolved in methanol (50 mL) and an excesssolution of HCl (4N solution in dioxane) (from Aldrich, 345547) (2 mL)added. The resulting solution was concentrated to dryness and the solidrecrystallized from ethylacetate/diethyl ether (1:1) to afford Cpd. 4 asa pale yellow solid (0.37 g., 24% yield).

¹HNMR (DMSO-d₆) δH (ppm); 3.52, 2.7H (s, CH₃), 3.67, 0.3H (s, CH₃),3.71, 2.7H (s, CH₃), 3.75, 0.3H (s, CH₃), 4.48, 0.8H (d, CH J=7.6 Hz),4.53, 0.2H (d, CH J=6.7 Hz), 4.57-4.63, 1H (m, CH), 7.3-7.41, 3H (m,Ar—H), 7.44-7.51, 2H (m, Ar—H), 7.64-7.72, 4H (m, Ar—H), 8.9, 3H (br s,NH₃ ⁺) (diastereoisomers); LCMS analysis (solvent MeCN/H₂O/0.1% HCO₂H,5-95% gradient/H₂O 2.5 min, 95% 6 min., Phenomenex C18 reverse phase,flow rate 1 mL/min.) HPLC retention time 6.05 min.; mass found 314(M+H).

(E) Synthesis of biphenyl-4-yl-(3,6-dioxo-piperazin-2-yl)-acetic acidmethyl ester (Cpd. 5)

A solution of Fmoc-Gly-OH (from Bachem) (1.66 g., 5.6 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (from Aldrich, 39391)(1.1 g., 5.7 mmol), 1-hydroxybenzotriazole (from Aldrich, 54804) (0.76g., 5.6 mmol) and triethylamine (0.4 mL, 2.8 mmol) in dimethylformamide(50 mL) was stirred at room temperature for 1 hour. A solution of2-amino-3-biphenyl-4-yl-succinic acid dimethyl ester hydrochloride (0.98g., 2.8 mmol) in dimethylformamide (10 mL) was added and the reactionmixture was stirred at room temperature for a further 14 hours. Thesolvent was removed in vacuo and the residue partitioned betweenethylacetate (100 mL) and sodium bicarbonate (100 mL, saturated solutionin water). The aqueous layer was washed with ethylacetate (2×100 mL),the organics combined, dried over magnesium sulphate and concentrated invacuo to afford a crude residue. The crude product was purified usingflash chromatography (SiO₂) eluting with ethylacetate/isohexane (3:1) toafford2-biphenyl-4-yl-3-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-acetylamino]-succinicacid dimethyl ester as a white crystalline solid (0.89 g., 54% yield).¹HNMR (CDCl₃) δH (ppm); 3.71-3.73 (m, 7H), 3.81 0.2H (d, CH J=5.6 Hz),3.86, 0.3H (d, CH J=5.8 Hz), 3.9, 0.3H (d, CH J=5.3 Hz), 3.94, 0.2H (d,CH J =5.8 Hz), 4.13-4.24 (m, 1H), 4.28-4.44 (m, 2.5H), 4.5 (d, 0.5HJ=4.4 Hz), 5.1-5.4 (m, 2H), 6.4 (d, NH J=8.5 Hz), 7.19 (d, NH J=9.4 Hz),7.23-7.34, 5H (m, Ar—H), 7.35-7.42, 4H (m, Ar—H), 7.47-7.60, 6H (m,Ar—H), 7.74, 2H (d, Ar—H J=7.6 Hz); LCMS analysis (solvent MeCN/H₂O/0.1%HCO₂H, 5-95% gradient/H₂O 2.5 min, 95% 4 min., Phenomenex C18 reversephase, flow rate 1 mL/min.) HPLC retention times 5.33 and 5.47 min.(diastereoisomers); mass found 593 (M+H).

2-Biphenyl-4-yl-3-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-acetylamino]-succinicacid dimethyl ester (0.86 g., 1.5 mmol) was dissolved in a solution of20% piperidine-dimethylformamide (30 mL) and the reaction mixture wasstirred at room temperature for 2 hours and then at 60° C. for 4 hours.The reaction mixture was concentrated in vacuo and the crude materialwas triturated using diethyl ether and then filtered to afford anoff-white solid. The solid material was triturated using ethylacetateand the isolated product washed with methanol, filtered and dried undervaccuum to afford Cpd. 5 as an off-white crystalline solid (0.26 g., 52%yield). ¹HNMR (DMSO-d₆) δH (ppm); 2.83, 1H (d, CH₂ J=17.2 Hz), 3.38,0.7H (dd, CH₂ J=2.9 & 17.2 Hz), 3.47, 0.3H (dd, CH₂ J=2.6 & 17.5 Hz),3.65, 3H (s, CH₃), 4.16, 0.3H (d, CH J=5.6 Hz), 4.27, 0.7H (d, CH J=4.7Hz), 4.43-4.46, 0.3H (m, CH), 4.5-4.53, 0.7H (m, CH), 7.3-7.37, 3H (m,Ar—H), 7.41-7.46, 2H (m, Ar—H), 7.59-7.68, 4H (m, Ar—H), 7.97-8.14, 2H(2×NH) (diastereoisomers); LCMS analysis (solvent 0.1% HCO₂H/MeCN,H₂O/0.1% HCO₂H, 7.5-95% gradient/H₂O 11 min, Thermo Betabasic 100×4.6 mmC18 reverse phase, flow rate 1 mL/min.) HPLC retention time 5.61 min.;mass found 339 (M+H).

Example 2 Inhibition of Proliferation of Cancer Cells

Assays were performed to determine the effect of Cpd. 5 (synthesisdescribed in Example 1) on proliferation of two cancer cell lines. Thesetwo cell lines were STTG grade 4 astrocytoma cell line and AU565 breastcancer cell line. Both cell lines were obtained from American TypeCulture Collection, Rockville, Md. (ATCC).

To perform the assays, a 50 mM stock solution of Cpd. 5 was prepared inDMSO and frozen at −80° C. Aliquots of the stock solution were removedand 100 μM dilutions were made in Iscove's modified Dulbecco's medium(IMDM; obtained from ATCC) supplemented with 10% fetal bovine serum(FBS) and penicillin/streptomycin solution. Controls containingequivalent amounts of DMSO to the 100 μM solution were mixed forcomparative purposes. 100 μl of the resulting solutions were then addedto 96 well culture plates in triplicate and placed in a 37° C. incubatorwith 5% CO₂ to warm. The controls were the PI3 kinase inhibitor,LY294002, and an inactive PI3 kinase inhibitor related compound,LY303511 (both obtained from Sigma).

At this point, cells were removed from passage flasks using trypsin andcounted using trypan blue to establish cell counts/ml and viability. Forall experiments, viability of cells was greater than 95%. Solutions of4,000 cells/100 μl were prepared in the medium from above and 100 μl ofthe solutions were added to each well of the plates. The plates wereplaced back in the incubator and incubated with the compounds for 96hours.

Following incubation, cell proliferation was assayed by adding 20 μlPromega cell titer aqueous one reagent to each well. Promega cell titerreagent is a solution containing a tetrazolium dye that is reduced byliving cells to a formazan dye, and this reduction is proportional tothe number of living cells present in the well. The plates wereincubated at 37° C. for an additional 4-18 hours to allow thecolorimetric assay to develop, and the OD of each well was determined ina microplate reader using a 530 nm filter. The OD of wells containingcell titer reagent in culture medium with no cells was subtracted fromthe OD of all experimental readings.

The % decrease of the total cell titer signal was determined using thefollowing formula. ((Diluent OD−Experimental OD)/Diluent OD))×100. Dataare presented in Table 1 as mean % decrease of cell titer signal ofthree separate experiments, plus or minus standard deviation. In themean % decrease column, a positive number means a decrease in cell titersignal, while a negative number means an increase in cell titer signal.The p values were calculated versus diluent controls.

TABLE 1 Mean % Decrease MEAN % STD. COMPOUND CELLS DECREASE DEVIATION PVALUE CPD. 5 STTG 78.74 3.93 0.000 (50 μM) LY294002 STTG −46.72 24.130.014 (20 μM) LY303511 STTG −4.74 6 0.121 (20 μM) CPD. 5 AU565 13.446.36 0.011 (50 μM) LY294002 AU565 61.95 6.58 0.000 (20 μM) LY303511AU565 −2.34 3.02 0.125 (20 μM)

To determine whether the decrease in cell titer signal caused by Cpd. 5was due to the inhibition of proliferation or to cytotoxicity, STTG andAU565 cells were seeded at 10,000 cells/well in 96-well plates and grownuntil a confluent monolayer was obtained. Under these conditions, thecells should no longer proliferate. The culture medium was IMDMsupplemented with 10% FBS. After the cells reached confluence, theculture medium was removed by aspiration and replaced with fresh mediumcontaining the test compounds (see Table 2 below). The cell cultureswere incubated for an additional 24 hours in the presence of the addedcompounds and then the cell titer signal was assayed by adding 20 μlPromega cell titer aqueous one reagent to each well. The plates wereincubated at 37° C. for an additional 2 hours to allow the colorimetricassay to develop, and the OD of each well was determined in a microplatereader using a 530 nm filter. The OD of wells containing cell titerreagent in culture medium with no cells was subtracted from the OD ofall experimental readings. The results of triplicate experiments arepresented in Table 2.

The % decrease of the total cell titer signal was determined using thefollowing formula. ((Diluent OD−Experimental OD)/Diluent OD))×100. Dataare presented in Table 2 as mean % decrease of cell titer signal ofthree separate experiments, plus or minus standard deviation. In themean % decrease column, a positive number means a decrease in cell titersignal, while a negative number means an increase. The p values werecalculated versus diluent controls.

This experiment was repeated, except using incubations of 18 hours,instead of 24 hours, in the presence of the compounds and 4 hours,instead of 2 hours, for color development. The results are presented inTable 3 below.

The combined results of the experiments show that the majority of thedecrease in cell titer signal caused by Cpd. 5 was due to inhibition ofproliferation and not to cytotoxicity.

TABLE 2 Mean % Decrease MEAN % STD. COMPOUND CELLS DECREASE DEVIATION PVALUE CPD. 5 AU565 −8.87 9.53 0.091 (10 μM) CPD. 5 AU565 −10.79 11.320.087 (50 μM) CPD. 5 AU565 −11.05 1.58 0.000 (100 μM) LY294002 AU5652.94 25.74 0.426 (50 μM) 70% Methanol* AU565 100.19 3.45 0.000 CPD. 5STTG −7.13 20.93 0.293 (10 μM) CPD. 5 STTG −1.76 21.08 0.446 (50 μM)CPD. 5 STTG −10.79 26.38 0.259 (100 μM) LY294002 STTG 26.12 13.10 0.013(50 μM) 70% Methanol* STTG 103.85 6.69 0.000 *Added 5 minutes prior tocell titer reagent.

TABLE 3 Mean % Decrease MEAN % COMPOUND CELLS DECREASE p VALUE CPD. 5AU565 −5.61 0.283 (10 μM) CPD. 5 AU565 −9.73 0.052 (50 μM) CPD. 5 AU565−10.69 0.038 (100 μM) LY294002 AU565 22.25 0.004 (50 μM) 70% Methanol*AU565 98.03 0.000 CPD. 5 STTG 0.81 0.313 (10 μM) CPD. 5 STTG 21.25 0.000(50 μM) CPD. 5 STTG 18.96 0.028 (100 μM) LY294002 STTG 26.27 0.001 (50μM) 70% Methanol* STTG 99.16 0.000 *Added 5 minutes prior to cell titerreagent.

Example 3 Inhibition of MMP-9

As a result of their ability to cleave components of the extracellularmatrix (ECM) and remodel the cellular microenvironment, matrixmetalloproteinases (MMPs) are thought to play a role in the developmentand progression of tumors. Thiennu H. Vu and Zena Werb, “Gelatinase B:Structure, Regulation, and Function,” pages 115-148, in MatrixMetalloproteinases (Academic Press, Editors William C. Parks and RobertP. Mecham, 1998). The type IV collagenases, in particular, have beenimplicated in tumor invasion and metastasis due to their ability todegrade basement membrane collagens. Id. Matrix metalloproteinase-9(MMP-9) (also known as gelatinase B) is widely thought to be a type IVcollagenase. Id. MMP-9, while not commonly expressed in normal cells,has been found to be expressed in tumors from diverse sites, includingskin, lungs, breast, colorectum, liver, prostate, brain, bone marrow andbone. Id. In some tumors, MMP-9 is expressed by the tumor cellsthemselves. Id. In other cases, other cells surrounding or found in thetumors express MMP-9. Id. In many tumors, high expression of MMP-9correlates with tumor invasiveness and metastatic potential. Id.

Assays were performed to determine the effect of Cpd. 5 (synthesisdescribed in Example 1) on the secretion of MMP-9 by the BT001 gliomacell line. The BT001 cell line was established as follows from cellsobtained after surgery. The excised tissue was treated briefly with aprotease cocktail, and the resulting cell suspension was cultured inIMDM supplemented with 10% FBS. Expanded cells were then frozen forfuture use.

A fresh 50 mM stock solution of Cpd. 5 was prepared in ethanol andwarmed to 37° C. Aliquots of the stock solution were removed and dilutedin IMDM supplemented with 0.1% FBS, insulin-transferrin-sodium selenite(ITSS) solution, and penicillin/streptomycin solution to prepareexperimental culture media (final concentrations of Cpd. 5 ranged from10-100 μM). Ethanol in the culture medium was used as diluent control.

The BT001 cells were cultured in 25 cm² flasks in IMDM containing 10%FBS at 37° C. and 5% CO₂. When cells reach 70-80% confluence, they werewashed two times with IMDM that had been warmed to 37° C. in a waterbath. The experimental culture media containing Cpd. 5 were then addedto the flasks (4.5 ml per flask), and the flasks were incubated at 37°C. and 5% CO₂ for 48 hours. The conditioned media were then removed fromthe cells, and cellular debris was removed by centrifugation at 2000 rpmfor 10 minutes. The supernatants were then transferred to sterile tubesand stored at −20° C.

Gelatin zymograms were used to evaluate MMP release and activity in thesamples. Precast 10% polyacrylamide gels impregnated with gelatin wereobtained from Invitrogen, Carlsbad, Calif. Ten μL of each of the thawedconditioned media were mixed with an equal volume of SDS-PAGE loadingdye without reducing agent (obtained from Invitrogen, Carlsbad, Calif.),applied to the gels and separated at 150 volts for 1 hour. The gels werethen processed following the manufacturer's recommended protocol(includes renaturing the gels for one hour and incubating overnight at37° C. with gentle shaking for protease digestion of gelatin).Visualization of MMP activity was done by staining the gel withInvitrogen Simple Safe blue commassie solution and photographing the gelon a Kodak Image station. The intensity of the bands in the photos wasdetermined using bundled Kodak software. Percent decrease was determinedwith the following formula:(Intensity of diluent control)−(Intensity of treatment)/(Intensity ofdiluent control)×100.The results are presented below in Table 4. The data presented in Table4 are the means of three or four replicate experiments.

TABLE 4 Mean % Decrease P VALUE MEAN % P VALUE MEAN % (VERSUS DECREASE(VERSUS DECREASE DILUENT ACTIVE DILUENT COMPOUND CELLS PRO-MMP-9CONTROL) MMP-9 CONTROL) Cpd. 5 BT001  6.89 ± 20.51 0.297 49.55 ± 21.490.008 (10 μM) Cpd. 5 BT001 36.13 ± 20.53 0.019 40.51 ± 9.73  0.002 (50μM) Cpd. 5 BT001 45.55 ± 20.50 0.297 71.08 ± 17.47 0.001 (100 μM)

The same experiment as described above for BT001 cells was performedusing STTG astrocytoma cells (for a description of the STTG cell line,see Example 2). It was found that Cpd. 5 had no effect on the release ofMMP-9 from the STTG cells (data not shown).

A similar experiment as described above for BT001 cells was performedusing primary human microglial cells (isolated from a lobectomy from anepileptic patient). It was found that 100 μM Cpd. 5 inhibited therelease of active MMP-9 from the primary microglial cells by 35.89%(using supernatants that had been concentrated 10×). Cpd. 5 did notinhibit the release of pro-MMP-9 from the primary microglial cells(using supernatants that had been concentrated 10×; data not shown).

Example 4 Inhibition of Proliferation of Cancer Cells

Assays were performed to determine the effect of Cpd. 5 on proliferationof an additional cancer cell line. This cell line was the U-118metastatic astrocytoma cell line. It was obtained from ATCC.

To perform the assays, a 50 mM stock solution of Cpd. 5 was prepared inDMSO and frozen at −80° C. Aliquots of the stock solution were removedand dilutions containing from 50 μM to 300 μM Cpd. 5 were made inIscove's modified Dulbecco's medium (IMDM; obtained from ATCC)supplemented with 10% fetal bovine serum (FBS) andpenicillin/streptomycin solution. Controls containing equivalent amountsof DMSO to the 300 μM solution were mixed for comparative purposes. 100μl of the resulting solutions were then added to 96 well culture platesin triplicate and placed in a 37° C. incubator with 5% CO₂ to warm. Thecontrols included the PI3 kinase inhibitor, LY294002, and an inactivePI3 kinase inhibitor related compound, LY303511 (both obtained fromSigma).

Cells were removed from passage flasks using trypsin andethylenediaminetetracetic acid (EDTA) and counted using trypan blue toestablish cell counts/ml and viability. For all experiments, viabilityof cells was greater than 95%. Solutions of 4,000 cells/100 μl wereprepared in the medium from above, and 100 μl of the solutions wereadded to each well of the plates. The plates were placed back in theincubator and incubated with the compounds for 96 hours.

Following incubation, cell proliferation was assayed by adding 20 μlPromega cell titer aqueous one reagent to each well. Promega cell titerreagent is a solution containing a tetrazolium dye that is reduced byliving cells to a formazan dye, and this reduction is proportional tothe number of living cells present in the well. The plates wereincubated at 37° C. for an additional 4 hours to allow the colorimetricassay to develop, and the OD of each well was determined in a microplatereader using a 530 nm filter. The OD of wells containing cell titerreagent in culture medium with no cells was subtracted from the OD ofall experimental readings. The results are presented in Table 5 below.

The % decrease of the total cell titer signal was determined using thefollowing formula. ((Diluent OD−Experimental OD)/Diluent OD))×100. Dataare presented in Table 5 as mean % decrease of cell titer signal ofthree separate experiments, plus or minus standard deviation. In themean % decrease column, a positive number means a decrease in cell titersignal, while a negative number means an increase in cell titer signal.The p values were calculated versus diluent controls.

TABLE 5 Mean % Decrease COMPOUND (Final Concentration) CELLS MEAN %DECREASE CPD. 5 U-118 65.84 (150 μM) CPD. 5 U-118 25.93 (100 μM) CPD. 5U-118 4.11 (75 μM) CPD. 5 U-118 −7.82 (50 μM) CPD. 5 U-118 −46.50 (37.5μM) CPD. 5 U-118 −50.62 (25 μM) LY294002 U-118 19.75 (20 μM) LY303511U-118 −25.93 (20 μM)

The above experiment was repeated using the U-118 cells at 3000cells/well. The results are shown in Table 6 below.

TABLE 6 Mean % Decrease COMPOUND (Final MEAN % Concentration) CELLSDECREASE P VALUE CPD. 5 U-118 59.70 0.003 (150 μM) CPD. 5 (100 μM) U-11820.51 0.108 CPD. 5 U-118 −3.05 0.404 (75 μM) CPD. 5 U-118 −15.63 0.177(50 μM) CPD. 5 U-118 −25.28 0.062 (37.5 μM) CPD. 5 U-118 −25.28 0.087(25 μM) LY294002 U-118 86.04 0.001 (40 μM) LY303511 U-118 0.41 0.490 (40μM)

Example 5 Effects of Cpd. 5 on HUVECs

Assays were performed to determine the effect of Cpd. 5 on proliferationof human umbilical vein endothelial cells (HUVECs). Passage 4-5 HUVECs,human source lot number 13047 (obtained from Lonza) were put into thewells of a 96-well tissue culture plate at 1200 cells/well in 100 μlendothelial growth medium-2 (EGM-2) complete medium (obtained fromLonza). A 50 mM stock solution of Cpd. 5 was prepared in DMSO and frozenat −80° C. Aliquots of the stock solution were removed and dilutionscontaining from 50 μM to 300 μM Cpd. 5 were made in EGM-2 completemedium (Lonza). Controls containing equivalent amounts of DMSO to the200 μM solution were mixed for comparative purposes. 100 μl of theresulting solutions were then added to the cells in the 96-well cultureplates in triplicate and placed in a 37° C. incubator with 5% CO₂ for 72hours. The controls were the PI3 kinase inhibitor, LY294002, and aninactive PI3 kinase inhibitor related compound, LY303511 (both obtainedfrom Sigma).

Following incubation, cell proliferation was assayed by adding 20 μlPromega cell titer aqueous one reagent to each well. Promega cell titerreagent is a solution containing a tetrazolium dye that is reduced byliving cells to a formazan dye, and this reduction is proportional tothe number of living cells present in the well. The plates wereincubated at 37° C. for an additional 4 hours to allow the colorimetricassay to develop, and the OD of each well was determined in a microplatereader using a 530 nm filter. The OD of wells containing cell titerreagent in culture medium with no cells was subtracted from the OD ofall experimental readings.

The % decrease of the total cell titer signal was determined using thefollowing formula. ((Diluent OD−Experimental OD)/Diluent OD))×100. Dataare presented in Table 7 as mean % decrease of cell titer signal ofthree separate experiments, plus or minus standard deviation. In themean % decrease column, a positive number means a decrease in cell titersignal, while a negative number means an increase in cell titer signal.The p values were calculated versus diluent controls. The results arepresented in Table 7 below.

As shown in Table 7, Cpd. 5 at 100 μM final concentration inhibited theproliferation of HUVECs. At lower concentrations, it appeared tostimulate proliferation of HUVECs. The reason for this stimulation isnot known, but may be due to the DMSO in which Cpd. 5 is dissolved.

TABLE 7 Mean % Decrease MEAN % COMPOUND DECREASE STD. DEVIATION P VALUECPD. 5 −48.60 20.79 0.040 (25 μM) CPD. 5 −14.44 17.99 0.234 (50 μM) CPD.5 62.04 1.54 0.000 (100 μM)

When endothelial cells are cultured on extracellular matrix protein gelsin the presence of angiogenic signals, they arrange themselves intostructures loosely resembling capillary blood vessels (“tubeformation”). Cpd. 5 was tested for its ability to affect tube formationand was found to have no observable effect at 50 μM or 100 μM.

Example 6 Inhibition of Akt Phosphorylation in Breast Cancer Cells

Assays were performed to determine the effect of Cpd. 5 (synthesisdescribed in Example 1) on phosphorylation of Akt in the AU565 breastcancer cell line. Akt (or protein kinase B, PKB) is a serine/threoninekinase that promotes cellular survival. Akt is activated in response tomany different growth factors, including IGF-1, EGF, bFGF, insulin,interleukin-3 (IL-3), IL-6, heregulin and VEGF. Akt has three isoforms,and activation of all three isoforms is similar in that phosphorylationof two sites is necessary for full activity. Once activated, Akt exertsanti-apoptotic effects through phosphorylation of substrates thatdirectly or indirectly regulate apoptosis. Activation of thephosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway contributesto tumorigenesis in many types of tissues, including breast, ovarian,brain, prostate and lymph tissues. It has been found that Akt isconstitutively active in an array of cancers and contributes to bothchemotherapeutic resistance and radiation resistance.

AU565 cells were grown in 75 cm² flasks using Iscove's modifiedDulbecco's medium (IMDM; obtained from Lonza) supplemented with 10%fetal bovine serum (FBS, HyClone) and 1% penicillin/streptomycinsolution (Lonza). Cells were removed from the flasks using trypsin/EDTA(Lonza) and counted using trypan blue to establish cell counts. Cells inthe above supplemented IMDM medium were added to the wells of a 96-wellplate (20,000 cells/well), and the plates were incubated at 37° C., 5%CO₂, for 24 hours. After 24 hours, the medium was removed and replacedwith medium not containing any serum (serum-free IMDM), and the plateswere incubated at 37° C., 5% CO₂, for an additional 24 hours.

After this incubation, the test compounds were added to the plates asfollows. A 20 mM stock solution of Cpd. 5 was prepared in DMSO andfrozen at −80° C. Aliquots of the stock solution were removed anddilutions were made in serum-free IMDM medium. Controls containingequivalent amounts of DMSO (0.05% and 0.25%) were mixed for comparativepurposes. The other control was the PI3 kinase inhibitor, LY294002(obtained from Sigma). 100 μl of each test compound solution were thenadded to 96 well culture plates in duplicate, and the plates were placedback into the incubator and incubated at 37° C., 5% CO₂, for 1 hour.

Following this incubation, 100 μl of serum-free IMDM containing either 0or 400 ng/ml insulin-like growth factor-1 (IGF-1) (obtained from Sigma),an Akt phosphorylation promoter, were added, and the plates incubated at37° C., 5% CO₂, for 1 more hour. At the end of this time, the cells werefixed immediately with 4% formaldehyde, refrigerated, and the extent ofphosphorylation of Akt determined using the Akt Cellular Activation ofSignaling ELISA (CASE™ Kit for AKT S473; SABiosciences, Frederick, Md.)following the manufacturer's protocols. The CASE™ Kit for AKT S473quantifies the amount of activated (phosphorylated) Akt protein relativeto total Akt protein in parallel assays using a conventional ELISAformat with colorimetric detection. The Akt phosphorylation site isserine 473 and is recognized by one of the antibodies used in the one ofthe two parallel assays to provide the measure of activated Akt protein.The other antibody used in the other parallel assay recognizes Akt toprovide the measure of total Akt protein. Both primary antibodies aredetected using a horseradish peroxidase-labeled secondary antibody.Addition of the manufacturer's Developing Solution for 10 minutes,followed by addition of the manufacturer's Stop Solution, produces theproduct which can be measured colorimetrically.

The results were calculated as follows. The treatment groups containingIGF-I/DMSO were subtracted from their respective IGF-1/DMSO only groups(positive control). This value was divided by the difference between therespective DMSO only group (negative control) and the respectivepositive control. This value represents the percent inhibition of Aktphosphorylation attributable to the inhibitor alone since thecontribution of DMSO is subtracted out.

The results are presented in Table 8. As can be seen from Table 8, Cpd.5 completely inhibited phosphorylation of Akt.

TABLE 8 Mean % Inhibition MEAN % INHIBITION OF Akt STD. TREATMENTPHOSPHORYLATION DEVIATION P VALUE CPD. 5 104.0 21.4 0.038 (10 μM) CPD. 592.0 26.7 0.044 (50 μM) LY294002 92.4 30.5 0.035 (50 μM)

Example 7 Inhibition of Akt Phosphorylation in Melanoma Cells

Assays were performed to determine the effect of Cpd. 5 (synthesisdescribed in Example 1) on phosphorylation of Akt in WM-266-4, ametastatic melanoma cell line obtained from ATCC. The WM-266-4 cellswere grown in 25 cm² flasks using IMDM medium (obtained from Lonza)supplemented with 10% fetal bovine serum (FBS, HyClone) and 1%penicillin/streptomycin solution (Lonza). Cells were removed from theflasks using trypsin/EDTA (Lonza) and counted using trypan blue toestablish cell counts. Cells in the above supplemented IMDM medium wereadded to the wells of a 96-well plate (10,000 cells/well), and theplates were incubated at 37° C., 5% CO₂, for 24 hours. After 24 hours,the medium was removed and replaced with medium not containing any serum(serum-free IMDM), and the plates were incubated at 37° C., 5% CO₂, foran additional 24 hours.

After this incubation, the test compounds were added to the plates asfollows. A 20 mM stock solution of Cpd. 5 was prepared in DMSO andfrozen at −80° C. Aliquots of the stock solution were removed anddilutions were made in serum-free IMDM medium. Final concentrations ofCpd. 5 were 10 μM and 50 μM. Controls containing equivalent amounts ofDMSO as the two concentrations of Cpd. 5 (0.05% and 0.25%) were mixedfor comparative purposes. The other control was the PI3 kinaseinhibitor, LY294002 (obtained from Sigma), final concentration of 50 μM.100 μl of each test compound solution were then added to 96 well cultureplates in duplicate, and the plates were placed back into the incubatorand incubated at 37° C., 5% CO₂, for 1 hour.

Following this incubation, 100 μl of serum-free IMDM containing either 0or 400 ng/ml IGF-1 (obtained from Sigma), an Akt phosphorylationpromoter, were added, and the plates incubated at 37° C., 5% CO₂, for 1more hour. At the end of this time, the cells were fixed immediatelywith 4% formaldehyde, refrigerated, and the extent of phosphorylation ofAkt determined using the Akt Cellular Activation of Signaling ELISA(CASE™ Kit for AKT S473; SABiosciences, Frederick, Md.) following themanufacturer's protocols. The CASE™Kit for AKT S473 quantifies theamount of activated (phosphorylated) Akt protein relative to total Aktprotein. For further details of the CASE ELISA assay, see Example 6.

The results were calculated as follows. The treatment groups containingIGF-I/DMSO were subtracted from their respective IGF-1/DMSO only groups(positive control). This value was divided by the difference between therespective DMSO only group (negative control) and the respectivepositive control. This value represents the percent inhibition of Aktphosphorylation attributable to the inhibitor alone since thecontribution of DMSO is subtracted out.

The results are presented in Table 9 below. As shown there, Cpd. 5 at 10μM gave 101.8% inhibition and at 50 μM gave 47% inhibition of Aktphosphorylation, as compared to 23.1% inhibition for 50 μM LY294002, theknown PI3 kinase inhibitor. It should also be noted that, in a separateexperiment, Cpd. 5 did not inhibit the proliferation of WM-266-4 cells,whereas LY294002 did inhibit proliferation of these cells (by 105%).

TABLE 9 Mean % Inhibition MEAN % INHIBITION OF Akt STD. TREATMENTPHOSPHORYLATION DEVIATION P VALUE CPD. 5 101.8 3.9 0.026 (10 μM) CPD. 555.9 21.7 0.036 (50 μM) LY294002 23.1 21.2 0.309 (50 μM)

In the WM-266-4 melanoma cell line there are two proliferationpathways—one through Akt kinase and one through MAP kinase (MAPK). SeeRusso et al., Int. J. Oncol., 34 (6):1481-1489 (June 2009) (abstract,PMID 19424565, full article in process) (in melanoma, both the MAPK andPI3K/Akt pathways are constitutively activated through multiplemechanisms). Both kinases are dependent on PI3 kinase, which wouldexplain why LY294002 inhibited proliferation of these cells, whereasCpd. 5 did not, although both compounds inhibited Akt phosphorylation.Even though Cpd. 5 did not inhibit proliferation of the WM-266-4 cellline, the inhibition of Akt phosphorylation (activation) by Cpd. 5should make the WM-266-4 cells more sensitive to standard anti-cancertherapies (e.g, chemotherapy and radiation). See Sinnberg et al., J.Invest. Dermatol., PMID 19078992, abstract of article Epub Dec. 11, 2008(inhibition of the PI3K/Akt pathway potently increases the sensitivityof melanoma cells to chemotherapy).

1. A diketopiperazine of the following formula or apharmaceutically-acceptable salt thereof:

wherein: R¹ is: (a) a side chain of an amino acid, wherein the aminoacid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid,2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine,norleucine, serine, homoserine, threonine, aspartic acid, asparagine,glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine,homoarginine, citrulline, phenylalanine, p-aminophenylalanine, tyrosine,tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamineor ornithine; (b) —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together withthe adjacent ring nitrogen forms proline or hydroxyproline; or (c) aderivative of a side chain of an amino acid, wherein the amino acid isone of those recited in (a), and the derivatized side chain has: (i) an—NH₂ group replaced by an —NHR³ or —N(R³)₂ group, wherein each R³ mayindependently be a substituted or unsubstituted alkyl, cycloalkyl,heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl; (ii) an —OHgroup replaced by an —O—PO₃H₂ or —OR³ group, wherein each R³ mayindependently be a substituted or unsubstituted alkyl, cycloalkyl,heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl; (iii) a—COOH group replaced by a —COOR³ group, wherein each R³ mayindependently be a substituted or unsubstituted alkyl, cycloalkyl,heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl; (iv) a —COOHgroup replaced by a —CON(R⁴)₂ group, wherein each R⁴ may independentlybe H or a substituted or unsubstituted alkyl, cycloalkyl,heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl; (v) an —SHgroup replaced by —S—S—CH₂—CH(NH₂)—COOH or —S—S—CH₂—CH₂—CH(NH₂)—COOH;(vi) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)— group; (vii) a—CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH group; and/or (viii) an Hwhich is attached to a carbon atom replaced by a halogen; R² has formulaII, III or IV:

wherein: each R⁵ is independently aryl, heteroaryl, alkyl, cycloalkyl,heterocycloalkyl, alkoxy, aryloxy, acyl, carboxyl, hydroxyl, halogen,amino, nitro, sulfo or sylfhydryl, wherein each alkyl is optionallysubstituted with hydroxyl, amino or sulfhydryl; n is from 0 to 5; and R⁶is hydrogen or lower alkyl.
 2. The diketopiperazine of claim 1 wherein:R¹ is: (d) a side chain of an amino acid, wherein the amino acid isglycine, alanine, valine, norvaline, α-aminoisobutyric acid,2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine,norleucine, serine, homoserine, threonine, aspartic acid, asparagine,glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine,homoarginine, citrulline, phenylalanine, p-aminophenylalanine, tyrosine,tryptophan, thyroxine or ornithine; or (e) a derivative of a side chainof an amino acid, wherein the amino acid is one of those recited in (d),and the derivative of the side chain is one of those recited in part (c)of claim 1; and R² has formula II or III.
 3. The diketopiperazine ofclaim 2 wherein R¹ is: (f) a side chain of an amino acid, wherein theamino acid is glycine, alanine, valine, norvaline, α-aminoisobutyricacid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,isoleucine, norleucine, serine, homoserine, threonine, aspartic acid,asparagine, glutamic acid, glutamine, lysine, hydroxylysine, arginine,homoarginine, citrulline or ornithine; or (g) a derivative of a sidechain of an amino acid, wherein the amino acid is one of those recitedin (f), and the derivative of the side chain is one of those recited inpart (c) of claim
 1. 4. The diketopiperazine of claim 3 wherein R¹ isthe side chain of glycine, alanine, valine, leucine or isoleucine. 5.The diketopiperazine of claim 1 wherein n is 1-3 and at least one R⁵ isan aryl, heteroaryl or aryloxy.
 6. The diketopiperazine of claim 5wherein R⁵ is an aryl or aryloxy.
 7. The diketopiperazine of claim 6wherein R⁵ is phenyl.
 8. The diketopiperazine of claim 6 wherein R⁵ isphenoxy.
 9. The diketopiperazine of claim 1 wherein n is 1 and R⁵ is inthe 4 (para) position on the ring.
 10. The diketopiperazine of claim 1wherein R² has formula II.
 11. The diketopiperazine of claim 1 whereinR² has formula III.
 12. The diketopiperazine of claim 1 wherein R⁶ ismethyl.
 13. The diketopiperazine of claim 1 which isbiphenyl-4-yl-(3,6-dioxo-piperazin-2-yl)-acetic acid methyl ester.
 14. Apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and an active ingredient, wherein the active ingredient is adiketopiperazine according to any one of claims 1-13 or apharmaceutically-acceptable salt or thereof.